|Year : 2016 | Volume
| Issue : 2 | Page : 65-71
Genetic variations in the growth arrest-specific 6 protein gene in patients with acute coronary syndrome
Mohamed M Elshafey MD , Jehan H Sabry, Omnia E Abdalla, Rania F Abdel Ghany
Department of Clinical and Chemical Pathology, Benha Faculty of Medicine, Benha, Egypt
|Date of Submission||07-Jul-2015|
|Date of Acceptance||11-Oct-2015|
|Date of Web Publication||1-Mar-2017|
Mohamed M Elshafey
Department of Clinical and Chemical Pathology, Faculty of Medicine, Benha University, Benha 13111
Source of Support: None, Conflict of Interest: None
Growth arrest-specific gene 6 (GAS6) encodes a vitamin K-dependent protein that regulates inflammation, angiogenesis, and atherosclerotic plaque formation. The level of GAS6 expression is associated with plaque stability and stroke. The role of GAS6 in cardiovascular disease, particularly in acute coronary syndrome (ACS), was explored. The study investigated the role of the single nucleotide polymorphism (c.834 + 7G >A) of GAS6 in ACS. The genotype frequencies for GG, AG, and AA, respectively, in patients with ACS were 57.1% (16/28), 28.6% (8/28), and 14.3% (4/28) and were 20% (4/20), 40% (8/20), and 40% (8/20) in the control group. The AA genotype and A allele were less frequent in patients with ACS than in controls (P < 0.05). Our study indicates that the AA genotype and A allele of the GAS6 gene relate to ACS and may have a protective role against ACS.
Keywords: acute coronary syndrome, growth arrest-specific gene 6, single nucleotide polymorphism
|How to cite this article:|
Elshafey MM, Sabry JH, Abdalla OE, Abdel Ghany RF. Genetic variations in the growth arrest-specific 6 protein gene in patients with acute coronary syndrome. Benha Med J 2016;33:65-71
|How to cite this URL:|
Elshafey MM, Sabry JH, Abdalla OE, Abdel Ghany RF. Genetic variations in the growth arrest-specific 6 protein gene in patients with acute coronary syndrome. Benha Med J [serial online] 2016 [cited 2017 Sep 19];33:65-71. Available from: http://www.bmfj.eg.net/text.asp?2016/33/2/65/201290
| Introduction|| |
The term acute coronary syndrome (ACS) refers to any group of clinical symptoms compatible with acute myocardial ischemia and covers the spectrum of clinical conditions ranging from unstable angina to non-ST-segment elevation myocardial infarction to ST-segment elevation myocardial infarction .
GAS6 protein is a vitamin K-dependent protein encoded by GAS6 gene and secreted by leukocytes and endothelial cells in response to injury. It is structurally related to the anticoagulant protein S; the two proteins have 44% amino acid identity .
GAS6 protein is also thought to act as a bridge between apoptotic cells and the phagocytes that ingest them. The growth arrest-specific gene 6 (GAS6) has a number of diverse functions and contributes to the regulation of cell survival, proliferation, migration, and adhesion .
GAS6 protein, the product of growth arrest-specific (GAS) gene 6, is a ligand for the tyrosine protein kinase receptors Axl, Tyro3, and Mer, whose signaling has been implicated in cell growth, survival, adhesion, and migration. Although a secreted human vitamin K-dependent protein with close structural similarity with protein S, GAS6 protein does not exhibit anticoagulant properties but rather may be an important regulator of vascular homeostasis and platelet signaling .
GAS6 protein signals through its receptor tyrosine kinases and appears to modulate platelet outside–in signaling through GP a (IIb) b (III), playing a key role in the perpetuation of platelet aggregates and clot retraction .
GAS6 protein is also implicated in foam cell formation and neointimal proliferation in response to vascular injury. Thus, GAS6 protein acts at key points in the pathophysiology of atherosclerosis and thrombosis – two processes implicated in most acute cardiovascular pathologies .
Previous studies have reported the genetic structure and allelic variability of the human GAS6 gene , indicating that there is an association between stroke and a single nucleotide polymorphism (c.834 + 7G >A) in intron 8 of the GAS6 gene. This suggests a potentially protective role of the AA genotype .
| Patients and Methods|| |
The present study was conducted on 60 individuals, 42 male and 18 female, attending the Cardiology Department of Benha University Hospital during the period from February 2013 to February 2014. They were divided into two groups: the patient group and the control group.
The patient group included 40 patients with coronary artery disease (CAD) with symptoms of cardiac ischemia that was verified by means of ECG, diagnostic coronary angiography, and elevated levels of biochemical markers (CK-MB and troponin I).
The mean age (±SD) was 50.8 years (±6.3). There were 28 male and 12 female patients who had been admitted to the coronary care unit. Only patients who had experienced their last chest pain within the previous 48 h were included in the study. Patients were further classified into three subgroups: the stable angina pectoris group (SAP), the unstable angina pectoris group (UAP), and the acute myocardial ischemia group (AMI).
The control group included 20 apparently healthy individuals of matched age and sex. Only individuals without a clinical history of cardiovascular disease (CVD), with a normal ECG, and having normal levels of cardiac biochemical markers were included. Patients with renal or hepatic diseases and patients with chest pain more than 48 h were excluded.
All procedures were performed in accordance with ethical standards and were approved by the ethical committee of Benha University, and written consent was obtained from each of the study participants.
Initial blood samples were collected from patients once admitted to the emergency department, and only those with confirmed diagnosis of CVD were included in the study.
- First blood sample: A volume of 7 ml of venous blood was collected under complete aseptic precautions and divided into three tubes:
- A volume of 1.8 ml of blood was added into citrated tubes, centrifuged immediately at 3000 rpm for 10 min, and assayed for prothrombin time.
- A volume of 2 ml of blood was added into EDTA tubes (EDTA anticoagulated blood) to separate genomic DNA from peripheral blood lymphocytes by use of blood genomic DNA extraction kits and then stored at −20°C until analysis.
- The rest of the samples were put into plain test tubes without anticoagulant and left to coagulate. After coagulation, the samples were centrifuged (at 3000 rpm for 15 min). The separated serum was divided into two aliquots:
- One was used for the immediate assay of liver function tests, kidney function tests, and cardiac enzymes (CK-MB and troponin I).
- The second aliquot was stored at −20°C for subsequent assay of high-sensitivity C-reactive protein (HS-CRP).
- Second blood sample:
The second venous blood samples (4 ml) were collected from the patients after overnight fasting under complete aseptic precautions into plain tubes without anticoagulant. The plain test tubes were left to coagulate. After coagulation, the samples were centrifuged (at 3000 rpm for 15 min). The separated serum was used for the assay of lipid profile and Fasting Blood Sugar. Hemolyzed samples were discarded.
A volume of 5 ml of blood was collected from apparently healthy individuals after overnight fasting under complete aseptic precautions and then handled, divided in the same way as the patient samples.
The CK-MB reagent contains an antibody that binds to the M subunit of CK in the serum sample, thereby inhibiting the activity of the M subunit. The remaining activity, corresponding to CK-B fraction activity, is measured according to the IFCC reference method for measuring CK activity. CK-MB activity is then obtained by multiplying by 2 the remaining activity.
Troponin I determination
Troponin I concentrations were measured on the mini VIDAS instrument using VIDAS Troponin I Ultra (TNIU) kit provided by bioMerieux Inc. (Durham, North Carolina, USA), for determination of human cardiac troponin I in human serum or plasma using the ELFA technique.
Measurement of high-sensitivity C-reactive protein using enzyme-linked immunosorbent assay
HS-CRP was measured with a STAT-FAX reader using Accu-bind enzyme-linked immunosorbent assay kits provided by Monbind Inc. (Lake Forest, California, USA). The kit was designed for determination of HS-CRP in human serum or plasma using microplate immunoenzymatic assay.
Single nucleotide polymorphism detection and genotyping
DNA extraction was carried out using GeneJET Whole-Blood Genomic DNA Purification Mini Kit provided by Thermo Fisher Scientific Inc., Lithuania. Samples were digested with proteinase K in the supplied lysis solution. The lysate was then mixed with ethanol and loaded onto the purification column, where the DNA binds to the silica membrane. Impurities were effectively removed by washing the column with the prepared wash buffers. Genomic DNA was then eluted under low ionic strength conditions with the elution buffer.
The purpose of a PCR is to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing.
The expression of intron 8 (c.834 + 7G >A) of GAS6 gene was determined using the following primers: GAS6E8F 5′-TTC CCT CAA GAA AGA GCC CG-3′and GAS6E8R 5′-TCT CAT CCC AAA CCT CCA CA -3′.
Detection of amplified PCR product by means of agrose gel electrophoresis
The amplification products were separated by means of agarose gel electrophoresis in 2% agarose gel stained with ethidium bromide and photographed under UV light.
Restriction fragment length polymorphism analysis to detect different genotypes
GAS6 genotypes were determined using specific restriction digestion enzyme) ALWN1) to digest PCR product covering exon 8 and part of intron 8 (481 bp), and it is used to differentiate between A and G alleles of GAS6 gene as follows: A allele is digested into two fragments (345 and 136 bp), but G allele remains uncut.
Statistical analysis was performed using the computer program SPSS (Statistical package for social science), version 16 (SPSS Inc., Chicago, IL, USA). Descriptive statistics were calculated for the data in the form of mean ± SD. Student's t-test was used to compare the mean of two groups of numerical (parametric) data.
| Results|| |
GAS6 intron 8 c.834 + 7G >A polymorphism in patients with acute coronary syndrome and SAP
To determine whether c.834 + 7G >A single nucleotide polymorphism is associated with an increased risk for CVD, all participants were genotyped, as shown in [Table 1] and [Figure 1]. It was found that the c.834+7G>A GG, AG, and AA genotype frequencies, respectively, in 40 patients with ACS were 55% (n = 22), 30% (n = 12), and 15% (n = 6) and in 20 controls were 20% (n = 4), 40% (n = 8), and 40% (n = 8). The GG genotype was the most prevalent and the AA genotype was less frequent in ACS patients [6 (15%) than in controls, 8 (40%)]. The A allele frequency was particularly low in patients with ACS.
|Table 1 Comparison between patients and controls groups as regards genotypes|
Click here to view
The GAS6 intron 8 c.834 + 7G >A single nucleotide polymorphism is associated with a decreased risk for cardiovascular disease
A significantly lower percentage of the AA genotype was observed in the UAP subgroup compared with the control group and in patients with SAP or AMI [Table 2] and [Figure 2]. In fact, the percentage of patients with UAP with the AA genotype [5.6% (1/18)] was lower than that in the control group [40% (8/20)]. These findings suggest that the AA allele may protect patients from UAP. Moreover, there was no significant difference in GAS6 genotype distribution in patients with SAP and AMI versus the control group (P > 0.05). The percentage of patients with SAP with the AA genotype [16.7% (2/12)] and the percentage of patients with AMI with the AA genotype [30% (3/10)] were slightly lower than that in the control group [40% (8/20)].
|Table 2 Comparison between patients' subgroups and controls as regards genotypes|
Click here to view
In the UAP group, patients with GG genotype had 24-fold greater risk than those with AA genotype, and the frequency of G allele was 3.8 times greater than that of A allele.
In the SAP group, patients with GG genotype had six-fold greater risk than those with AA genotype, and the frequency of G allele was 2.2 times greater than that of A allele. In the AMI group, patients with GG genotype had 2.7 times greater risk than those with AA genotype, and the frequency of G allele was 1.6 times greater than A allele. In the CAD group, patients with GG genotype had 7.3 times greater risk than those with AA genotype, and the frequency of G allele was 2.5 times greater than that of A allele [Table 3].
|Table 3 Odds ratio and confidence interval for genotypes and frequencies in controls, patients with stable angina pectoris, and patients with acute coronary syndrome|
Click here to view
| Discussion|| |
The present study was designed to evaluate the role of the single nucleotide polymorphism (c.834 + 7G >A) of GAS6 in CVD, particularly in ACS.
In the present study, hypertension was the most common risk factor for ACS (61.7%), followed by smoking (53.4%) and diabetes mellitus (43.3%). In a similar study, hypertension was the most common risk factor for ACS, followed by cigarette smoking and diabetes mellitus .
Several studies have reported smoking as a risk factor for ACS, followed by hypertension and diabetes mellitus, which differs from the results of the present study, probably because all female patients were nonsmokers ,,.
Dyslipidemia was one of the nine major risk factors (smoking, diabetes, hypertension, visceral obesity, psychosocial stress, sedentary life, low fruit and vegetable consumption, and alcohol consumption), and dyslipidemia alone accounted for more than 50% of population attributable risk .
Assessment of lipid profile parameters revealed that there was a statistically significant elevation in serum levels of total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), triglycerides (TG), in ACS patients compared with controls, whereas high-density lipoprotein-cholesterol (HDL-C) was significantly low in ACS patients compared with controls (P < 0.001).
A similar study reported that dyslipidemia, manifested by elevated levels of TC and LDL-C, low levels of HDL-C, and high levels of TG, is an important risk factor for CAD .
Moreover, several studies reported increased TC, TG, and LDL-C and decreased HDL-C levels in patients with ACS compared with controls ,.
Data from the present study revealed that there was no significant difference between patients with myocardial infarction (MI), UAP, and SAP as regards all lipid profile parameters; this is contradictory to the concept that low levels of HDL-C are significantly low in participants with MI compared with those with UAP ,.
CK-MB normally exists in the cellular compartment and leaks out into the plasma during myocardial injury due to disintegration of contractile elements and sarcoplasmic reticulum. Troponin I is a protein of the troponin regulatory complex involved in cardiac contractility. It has very high myocardial tissue specificity, not detectable in the blood of healthy individuals, and offers an improved sensitivity and specificity for AMI versus a combination of ECG and traditional biochemical markers .
The results of the present study showed a statistically highly significant increase in troponin I and CK-MB mean levels in patients with ACS versus controls. These results were in agreement with the results for CK, CK-MB, and troponin T in the study by Pasupathi et al. , and troponin I levels were significantly increased (P < 0.001) in individuals suffering from MI and Ischaemic heart disease compared with controls.
A subgroup analysis revealed that there was a statistically significant increase in the mean troponin I and CK-MB concentration in patients with AMI versus the UAP group (P1< 0.05), and AMI versus the SAP group (P2< 0.05), but there was no statistically significant difference in the mean troponin I and CK-MB concentration between the UAP group and the SAP group (P3> 0.05); this is in agreement with the fact that patients with unstable angina and AMI had significantly higher concentrations of troponin I and CK-MB compared with patients with stable angina or controls .
A similar study found that serum TnT and CK-MB levels were also significantly high in AMI patients as compared with controls .
A possible explanation for elevated levels of CK-MB and troponin I is myocardial necrosis .
The degree of coronary obstruction may affect the degree of myocyte injury and hence the troponin I and CK-MB levels. An occlusive thrombus in the absence of significant collateral vessels most often results in ST-segment elevation myocardial infarction. Angiographic studies had shown that evidence of thrombus, complex lesions, and reduced TIMI (Thrombolysis in Myocardial Infarction) flow grade were more common in patients with elevated troponin levels than in those with normal levels .
HS-CRP is a positive acute phase protein synthesized by the liver cells; its level rises in response to inflammation .
HS-CRP release is triggered by various proinflammatory stimulants such as cytokines, oxidized LDL, and infectious agents .
In patients with CAD, increased level of HS-CRP is regarded as an important prognostic indicator for risk stratification in acute and recurrent attack, as it directly and actively participates in both atherogenesis and atheromatous plaque disruption .
Many studies described that elevation of serum CRP levels is related to increased risk for MI ,.
CRP induces activation of peripheral leucocytes with subsequent secretion of plaque-destabilizing mediators. These findings are consistent with the hypothesis that infectious diseases trigger manifestations of atherosclerosis, in which CRP elevation might contribute to the onset of cardiovascular events.
The earliest research from Italy found that elevated CRP predicts a poor outcome in patients with unstable angina .
There was a highly significant difference between patients and controls as regards HS-CRP level, and this is supported by another study, which demonstrated a higher level of HS-CRP in patients with ACS versus controls .
Moreover, in a subgroup analysis, there was a significant increase in the mean HS-CRP concentration in patients with AMI versus the UAP group (P1< 0.05) and AMI versus the SAP group (P2< 0.05) as well as in patients with UAP versus the SAP group (P3< 0.05) and this is supported by another study, which found significant elevation in all patients .
This is in disagreement with another study, which demonstrated that HS-CRP level was not significantly increased in patients with unstable angina .
There was a significant difference between patients and controls as regards GAS6 genotypes; AA genotype was less frequent in cases versus controls (15 vs. 40%, respectively; P < 0.05). In contrast, the GG genotype was more frequent in cases versus controls (55 vs. 20%, respectively; P < 0.05).
A similar study found that the AA genotype was expressed at a lower frequency in patients with ACS compared with patients with SAP, which may suggest a protective role for this GAS6 variant in ACS and indicates that GAS6 may be a candidate susceptibility gene for this disease .
Therefore, GAS6 polymorphisms that would result in differences in GAS6 protein levels could influence the regulation of atherogenesis or the activation of endothelial cells and vascular smooth muscle cells by this protein.
Another study stated that GAS6 is associated with CVD and provides further evidence that the AA genotype of the c.834 + 7G >A GAS6 polymorphism may have a protective role against ACS .
A subgroup analysis of the present study as regards AA, AG, and GG genotypes revealed that there was no statistically significant difference between patients with SAP and with AMI versus controls (P > 0.05). This is supported by another study, which found no difference in allelic frequency and genotype distribution of GAS6 gene between the AMI and control groups, with a similar distribution of GG, GA, and AA genotypes .
Hence, the current study revealed that the GAS6 c.834 + 7G >A polymorphism is associated with a lower risk for CVD. The G allele of GAS6 gene is the risky allele, which is significantly more frequent in patients with ACS than in controls. The results also suggest a protective role of the AA genotype.
| Conclusion|| |
The different GAS6 genotypes (GG, AG, and AA) were all expressed in patients with CVD. The expression of the A allele was lower in patients with ACS than in patients with SAP and controls. The results also indicate that the GAS6 c.834 + 7G >A polymorphism is associated with a lower risk for CVD, particularly for the subtypes affecting atherosclerotic plaque destabilization.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Lloyd-Jones D, Adams R, Carnethon M, et al.
American Heart Association Statistics Committee and Stroke Statistics Subcommittee Heart disease and stroke statistics–2009 update. A report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2009; 119:480–486.
Carl E, Adam L, Per Å. Plasma concentrations of Gas6 (growth arrest specific protein 6) and its soluble tyrosine kinase receptor sAxl in sepsis and systemic inflammatory response syndromes. Crit Care 2010; 14:R158.
Gibot S, Massin F, Cravoisy A, et al.
Growth arrest-specific protein 6 plasma concentrations during septic shock (2007). Crit Care; 11: R8 Gas6 levels elevated in renal failure 4171. Available at: http://ndt.oxfordjournals.org/
. [Accessed 25 December 2014].
Maree AO, Jneid H, Fitzgerald DJ. Growth arrest specific gene (GAS) 6 modulates platelet thrombus formation and vascular wall homeostasis and represents an attractive drug target. Curr Pharm Des 2007; 13:2656–2661.
Angelillo-Scherrer A, Burnier L, Matsushima GK, et al.
Role of Gas6 receptors in platelet signaling during thrombus stabilization and implications for antithrombotic therapy. J Clin Invest 2005; 115:237–246.
Muñoz X, Sumoy L, Ramírez-Lorca R, et al.
Human vitamin K-dependent GAS6: gene structure, allelic variation, and association with stroke. Hum Mutat 2004; 23:506–512.
Muñoz X, Obach V, Hurtado B, et al.
Association of specific haplotypes of GAS6 gene with stroke. Thromb Haemost 2007; 98:406–412.
Saito Y, Kita T, Mabuchi H. Obesity as a risk factor for coronary events in Japanese patients with hypercholesterolemia on low-dose simvastatin therapy. J Atheroscler Thromb 2010; 17:270–277.
Cooke C, Hammerash W. Retrospective review of sex differences in the management of dyslipidemia in coronary heart disease: an analysis of patient data from a Maryland-based health maintenance organization. Excerpra Medica 2006; 28:591–599.
Lahoz C, Mostaza J, McConnell JP. Atherogenic dyslipidemia in patients with established coronary artery disease. Nutr Metab Cardiovasc Dis 2012; 22:103e108.
Sandhu K, Singh A, Nadar S. Management of elderly patients with troponin positive chest pain in a District General Hospital. Cardiol J 2012; 19:395–401.
Yusuf S, Hawken S, Lanas F, et al.
Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case–control study. Lancet 2004; 364:937−954.
Yadav AS, Bhagwat VR. Lipid profile pattern in anginal syndrome patients from Marathwada Region of Maharashtra State. J Med Edu Res 2012; 2:12–15.
Kamariya CP, Gorasia JH, Gohel M. Evaluation of serum lipoprotein (a) in young patients with myocardial infarction. Int J Med Public Health 2014; 4:107–109.
Esteghamati A, Abbasi M, Nakhjavani M. Prevalence of diabetes and other cardiovascular risk factors in an Iranian population with acute coronary syndrome. Cardiovasc Diabetol 2006; 5:15.
Guler E, Gecmen C, Karaca1 O. Adding lipoprotein(a) levels to the GRACE score to predict prognosis in patients with non-ST elevation acute coronary syndrome. KardiologiaPolska 2013; 71:695–701.
Kasap S, Gonenç A, Sener DE, et al.
Serum cardiac markers in patients with acute myocardial infarction: oxidative stress, C-reactive protein and N-terminal probrain natriuretic peptide. J Clin Biochem Nutr 2007; 41:50–57.
Pasupathi P, Rao Y, Farook J, et al.
Effect of cigarette smoking on lipids and oxidative stress biomarkers in patients with acute myocardial infarction. Res J Med Med Sci 2009; 4:151–159.
Garlichs CD, Eskafi S, Raaz D, et al.
Patients with acute coronary syndromes express enhanced CD40 ligand/CD154 on platelets. Heart 2001; 86:649–655.
Yilmaz A, Yalta K, Turgut O, et al.
Clinical importance of elevated CK-MB and troponin I levels in congestive heart failure. Adv Ther 2006; 23:1060–1067.
Diderholm E, Andron B, Frostfeldt G, et al.
The prognostic and therapeutic implications of increased troponin T levels and ST depression in unstable coronary artery disease: the FRISC II invasive troponin T electrocardiogram substudy. Am Heart J 2002; 143:760–767.
Napoli M, Elkind M, Popa-Wagner A, et al.
Role of C-reactive protein in cerebrovascular disease. Expert Rev Cardiovasc Ther 2011; 9:1565–1584.
Jialal I, Devaraj S. Inflammation and atherosclerosis the value of the high-sensitivity C-reactive protein assay as a risk marker, Am J Clin Pathol 2001; 116:108–115.
Sheikh A, Yahya S, Sheikh N, Sheikh N. C-reactive protein as a predictor of adverse outcome in patients with acute coronary syndrome, Heart Views 2012; 13:7–12.
Ramasamy I. Biochemical markers in acute coronary syndrome. Clin Chim Acta 2011; 412:1279–1296.
Windgassen EB, Funtowicz L, Lunsford TN. C-reactive protein and high-sensitivity C-reactive protein: an update for clinicians. Postgrad Med 2011; 123:114–119.
Bisoendial RJ, Birjmohun RS, van 't Veer C, et al.
C-reactive protein elicits white blood cell activation in humans. Am J Med 2009; 122:582.
Liuzzo G, Biasucci LM, Gallimore JR, et al.
The prognostic value of C-reactive protein and serum amyloid a protein in severe unstable angina. N
Engl J Med 1994; 331:417–424.
Hassan AM, Saleh ES, Sarkis K. Evaluation of fetuin – a protein and some inflammatory biomarkers in patients with coronary artery disease Am J Pharmacol Sci 2014; 2:87–92.
Fisher PW, Burke MB, Wu SJ, et al.
A novel site contributing to growth-arrest-specific gene 6 binding to its receptors as revealed by a human monoclonal antibody. Biochem J 2005; 387:727–735.
Jambou D, Candito M, Staccini P. AA genotype of single-nucleotide polymorphism c.834 + 7G >A in GAS6 gene (rs8191974) is not protective for acute myocardial infarction (2013). Blackwell Publishing Ltd Int. Jnl. Lab. Hem
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]